The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells 1 – 3 . Considerable effort has been invested using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary “high affinity” receptor complex consisting of IL-2, IL-2Rα (termed CD25), IL-2Rβ, and γ c 4 – 8 . Naïve T cells express only a low density of IL-2Rβ and γ c , and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ andγ c . Here, using in vitro evolution, we eliminated IL-2’s functional requirement for CD25 expression by engineering an IL-2 “superkine” (termed super-2) with increased binding affinity for IL-2Rβ. Crystal structures of super-2 in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, including a flexible helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in super-2 recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation T cells irrespective of CD25 expression. Compared to IL-2, super-2 induced superior expansion of cytotoxic T cells, leading to improved anti-tumor responses in vivo , and elicited proportionally less expansion of T regulatory cells and reduced pulmonary edema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy.

Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rβ into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4+ and CD8+ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.

We have developed a multiplexed reverse phase protein (RPP) microarray platform for simultaneous monitoring of site-specific phosphorylation of numerous signaling proteins using nanogram amounts of lysates derived from stimulated living cells. We first show the application of RPP microarrays to the study of signaling kinetics and pathway delineation in Jurkat T lymphocytes. RPP microarrays were used to profile the phosphorylation state of 62 signaling components in Jurkat T cells stimulated through their membrane CD3 and CD28 receptors, identifying a previously unrecognized link between CD3 crosslinking and dephosphorylation of Raf-1 at Ser259. Finally, the potential of this technology to analyze rare primary cell populations is shown in a study of differential STAT protein phosphorylation in interleukin (IL)-2-stimulated CD4(+)CD25(+) regulatory T cells. RPP microarrays, prepared using simple procedures and standard microarray equipment, represent a powerful new tool for the study of signal transduction in both health and disease.

Type 1 diabetes (T1D) may result from a breakdown in peripheral tolerance that is partially controlled by peripheral tissue antigen (PTA) expression in lymph nodes. Here we show that the transcriptional regulator deformed epidermal autoregulatory factor 1 (Deaf1) controls PTA gene expression in the pancreatic lymph nodes (PLN). The expression of canonical Deaf1 was reduced, while that of an alternatively spliced variant was increased during the onset of destructive insulitis in the PLN of NOD mice. An equivalent variant Deaf1 isoform was identified in the PLN of T1D patients. Both NOD and human Deaf1 variant isoforms suppressed PTA expression by inhibiting the transcriptional activity of canonical Deaf1. Reduced PTA expression resulting from the alternative splicing of Deaf1 may contribute to T1D pathogenesis.

Engagement of the IFN-αβ receptor initiates multiple signaling cascades, including activation of the STAT. In this study, we demonstrate that IFN-αβ, although antiproliferative in wild-type CD4+ or CD8+ T cells, act as strong mitogens on their STAT1−/− counterparts. Furthermore, IFN-αβ exert little effect on apoptosis in wild-type cells, but are potent survival factors in the absence of STAT1. The antiapoptotic response in the absence of STAT1 is predominantly mediated by STAT3, and to a lesser extent by STAT5A/B. In contrast, the mitogenic IFN-αβ response gained through the absence of STAT1 is only marginally affected when STAT5A/B expression is also abrogated, but is completely dependent on STAT3 activation. These findings provide the first evidence for a function of STAT3 and STAT5A/B in the IFN-αβ response, and support a model in which the IFN-αβ receptor initiates both pro- and antiapoptotic responses through STAT1, and STAT3 and STAT5A/B, respectively.

Type I and II interferons (IFNs) exert opposing effects on the progression of multiple sclerosis, even though both IFNs use the signal transducer and activator of transcription 1 (STAT1) as a signaling mediator. Here we report that STAT1-deficient mice expressing a transgenic T cell receptor against myelin basic protein spontaneously develop experimental autoimmune encephalomyelitis with dramatically increased frequency. The heightened susceptibility to this autoimmune disease appears to be triggered by a reduced number as well as a functional impairment of the CD4+ CD25+ regulatory T cells in STAT1-deficient animals. Adoptive transfer of wild-type regulatory T cells into STAT1-deficient hosts is sufficient to prevent the development of autoimmune disease. These results demonstrate an essential role of STAT1 in the maintenance of immunological self-tolerance.

Acquisition of the anergy phenotype in T cells is blocked by inhibitors of protein synthesis and calcineurin activity, suggesting that anergic T cells may have a unique genetic program. Retroviral transduction of hemopoietic stem cells from TCR transgenic mice and subsequent reconstitution of syngeneic mice to express the E3 ubiquitin ligase, gene related to anergy in lymphocytes (GRAIL), or an enzymatically inactive form, H2N2 GRAIL, allowed analysis of the role of GRAIL in T cell anergy in vivo. Constitutive expression of GRAIL was sufficient to render naive CD4 T cells anergic, however, when the enzymatically inactive form H2N2 GRAIL was expressed, it functioned as a dominant negative of endogenous GRAIL and blocked the development of anergy. These data provide direct evidence that a biochemical pathway composed of GRAIL and/or GRAIL-interacting proteins is important in the development of the CD4 T cell anergic phenotype in vivo.

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